Lipoprotein(a) [Lp(a)] was described as a genetic variant of low density lipoprotein (LDL) in 1963 (K. Berg (1963) Acta Pathol Microbiol Scand 59: 369-381). Later it was discovered that although Lp(a) resembles LDL in having similar lipid composition and a common apolipoprotein B-100 (apo B), Lp(a) contains an additional glycoprotein, named apolipoprotein(a) [apo(a)]. Each Lp(a) molecule contains one molecule of apo(a) per one molecule of apo B covalently linked by a sulfide bond that can be easily reduced to LDL and apo(a) (Gaubatz et al. (1983) J. Biol Chem 258: 4582-4589; Fless et al. (1984) J. Biol Chem 259: 11470-11478; Fless et al. (1986) J Biol Chem 261: 8712-8718, Fless et al (1994) Biochemistry 33: 13492-13501; Marcovina and Morrisett (1995) Curr Opin in Lipidology 6: 136-145; Albers et al. (1996) J Lipid Res 37: 192-196).
Lipoprotein(a) particles exhibit considerable inter- and intra-individual heterogeneity, with some individuals exhibiting two or more distinct Lp(a) particles differing in hydrated density (Fless et al. (1984) J Biol Chem 259: 11470-11478). Also, the Lp(a) particle varies widely in size, with the size heterogeneity related primarily to the size of the apo(a) isoforns, ranging from 280 to 838 KDa; to date, 34 different isoforms have been identified (Marcovina et al. (1993) Biochem Biophys Res Commun 191: 1192-1196). The number of apo(a) isoforms that can be distinguished varies from six to at least twelve isoforms. The smaller isoforms are generally present at less frequency and are associated with the higher Lp(a) concentrations, whereas the larger isoforms have a higher frequency and are associated with lower Lp(a) concentrations. There appears to be an inverse relationship between the apparent molecular mass of the apo(a) isoforms and the concentrations of Lp(a) in plasma (G. Utermann (1989) Science 246: 904-910; Morrisett et al. (1990) in Lipoprotein(a), Academic Press, pp. 53-74; Sandholzer et al. (1992) Arteriosclerosis and Thrombosis 12: 1212-1226).
The structural gene for apo(a) is located on chromosome 6 near the plasminogen gene (Frank et al. (1988) Hum Genet 79: 352-356). Sequencing of apo(a) at both the protein and cDNA level has revealed a high degree of homology to plasminogen (Eaton et al. (1987) Proc Natl Acad Sci 84: 3224-3228; McLean et al. (1987) Nature (London) 330: 132-137). Apo(a) contains two types of plasminogen-like domains: a single kringle 5 domain, with 82% amino acid sequence homology and 91% nucleotide sequence homology with plasminogen, and multiple repeats of a kringle 4 domain, with 61-75% amino acid homology and 75-85% nucleotide sequence homology with the kringle 4 domain of plasminogen. Homology to plasminogen is also revealed by immunochemical studies that show cross-reactivity of apo(a) and plasminogen (Karadi et al. (1988) Biochim Biophys Acta 960: 91-97); Lafferty et al. (1991 ) J Lipid Res 32: 277-292).
Numerous studies have indicated that elevated levels of Lp(a) in plasma are associated with premature coronary heart disease (CHD) (Scanu and Fless (1990) J Clin Invest 85: 1709-1715; Sandholzer et al. (1992) Arteriosclerosis and Thrombosis 12: 1214-1226; Seed et al. (1990) New Engl J Med 332: 1494-1499; Genest et al. (1992) J Am Coll Cardiol 19: 792-802; Dahlen et al. (1986) Circulation 74: 758-765). Lp(a) concentrations in human plasma range from 1 mg/dL to more than 100 mg/dL. When the plasma Lp(a) level is above 30 mg/dL, the relative risk of CHD is raised about two-fold. When LDL and Lp(a) are both elevated, the relative risk is increased to about five-fold (Armstrong et al. (1986) Atherosclerosis 62: 249-257). Recent studies have suggested that increased Lp(a) concentrations may inhibit fibrinolysis by reducing the generation of plasmin by competing for plasminogen cell-surface receptors, or inhibiting activation of plasminogen, or competing for binding sites on fibrin (Hajjar et al. (1989) Nature (London) 339: 303-305; Miles et al. (1989) Nature (London) 339: 301-303; Gonzalez-Gronow et al. (1989) Biochemistry 28: 2374-2377; Edelberg et al. (1989) Biochemistry 28: 2370-2374; Loscalzo et al. (1990) Anteriosclerosis 10: 240-245; Harpel et al. (1989) Proc Natl Acad Sci USA 86: 3847-3851; Angles-Cano (1994) Chem Phys Lipids 67/68: 353-362; 369-380; Liu et al. (1994) Biochemistry 33: 2554-2560; Hajjar and Nachman (1996) Annu Rev Med 47: 423-442).
More recently, it has been shown that the binding activity of the macrophage Lp(a)/apo(a) receptor can be blocked by a monoclonal antibody directed against a specific kringle 4 domain (subtypes 6-7) (Keesler et al (1996) J Biol Chem 27: 32096-32104). This suggests a possible role of Lp(a) in Lp(a)-induced atherogenesis. While the function of Lp(a) is unknown, a significant correlation has been established between elevated levels of Lp(a) and coronary artery and cardiovascular disease that led many scientists to study the physiological role of Lp(a) in heart disease (R. M. Lawn (1992) Scientific American pp. 54-60; Simon et al. (1993) Curr Opin in Lipidology 8: 814-820; Klezovitz and Scanu (1995) Curr Opin in Lipidology 6: 223-228; Durrington (1995) Bailliere Clin Endocrinol 9: 773-795).
A number of assay methods for quantitating Lp(a) in plasma are known (see Morrisett et al. (1987) in Plasma Lipoproteins, Elsevier Science B. V., Chapter 5, pp. 129-152; Gaubatz et al. (1986) in Methods in Enzymology, Vol. 129, pp. 167-187; Albers et al. (1990) Clin Chem 36: 2019-2026; Labeur and Rosseneu (1992) Curr Opin in Lipidology 3: 372-376; Albers and Marcovina (1994) Curr Opin in Lipidology 5: 417-421). The assays include radioimmunoassays, enzyme-linked immunosorbent assays (ELISAs), radial immunodiffusion, electroimmunoassays, immunoelectrophoresis and turbidimetric assays. Most of the Lp(a) assay methods except the ELlSAs are not commonly used due to inherent technical problems (Labeur and Rosseneu (1992) Curr Opin in Lipidology 3: 372-376). ELlSAs that are presently known use either monoclonal or affinity-purified polyclonal antibodies. The majority of the monoclonal antibodies recognize the kringle 4 epitope of apo(a), whereas the polyclonal antibodies recognize both kringle 4 and kringle 5 epitopes of apo(a) (Lafferty et al. (1991) J Lipid Res 32: 277-292; Fless et al. (1989) J Lipid Res 30: 651-662; Rainwater and Manis (1988) Atherosclerosis 73: 23-31).
As noted above, apo(a) contains multiple copies of kringle 4 domain. The multiple copies of apo(a) kringle 4 are similar but not identical to each other and can be divided into 10 distinct kringle types (kringle 4 types 1 through 10). One copy each of kringle 4 type 1 and types 3 through 10 is present per apo(a) molecule; kringle 4 type 2, however, is present in a variable number of repeats (from 3 to &gt;40) and are therefore responsible for the size heterogeneity of apo(a) and consequently Lp(a) (Lackner et al. Hum Molec Genet (1993) 2: 933-940; Van der Hoek et al. Hum Molec Genet (1993) 2: 361-366). From the structural sequence of kringle 4 repeats it seems obvious that the immunoreactivity of the antibodies used in the immunoassays to measure Lp(a) concentrations will vary according to the number of epitopes available in a particular Lp(a). Therefore, antibodies against apo(a) should be selected to be specific for that part of the apo(a) molecule that is independent of size polymorphism, i.e. for kringle 4 domains other than type 2 or kringle 5 domain.
Among the numerous papers published to date, only one reports the domain specificity of the monoclonal antibodies used in the immunoassays to measure Lp(a) (Marcovina et al. (1995) Clin. Chem 41: 246-255). Recently, an immunoassay method for the detection of Lp(a) was disclosed using an anti-apo(a) monoclonal antibody that was described as non-reactive with plasminogen and the kringle 4 type 2 repeats of apo(A) (see WO96/19500 published Jun. 27, 1996). Although Albers, Rosseneau, and others have suggested that an optimal antibody should be the one that is directed towards an epitope that is localized in the non-repetitive and non-glycosylated kringle 5 domain (Albers et al. (1990) Clin Chem 36: 2019-2026; Labeur and Rosenau (1992) Curr Opin in Lipidology 3: 372-376; Albers and Marcovina (1994) Curr Opin in Lipidology 5: 417-421), it was not been possible until recently to develop kringle 5 domain specific antibodies because of extensive problems associated with generating domain specific antibodies.
A polyclonal antibody was recently developed by immunizing a sheep with a cloned kringle 5 fusion protein (Chenivesse et al. (1996) Protein Expression and Purification 8: 145-150). This antibody was shown by ELISA and Western blot to react with Lp(a) and the C-terminal domain of apo(a), but not with the kringle 4 repeats at the N-terminal end. In both formats, the proteins were immobilized on solid phases, sometimes under denaturing conditions. No data was provided on whether this polyclonal antibody cross-reacted with plasminogen or any of the other lipoproteins that are abundant in human plasma.
The reactivity of an antibody for its specific antigen can differ considerably depending on the type of assay format it is used in, i.e. how and where in the assay the antibody is utilized. The state of the antigen, e.g. whether it is in solution or attached to a solid phase, how it is attached to a solid phase, whether it is denatured or not, also affects antibody binding; some antibodies recognize conformation-dependent epitopes and therefore require the antigen to be in its native state. Moreover, the specificity and immunoreactivity of polyclonal antibodies can vary from animal to animal and species to species making it difficult to produce a reliable and consistent immunoassay. Therefore, monoclonal antibodies are presently needed which are specific for an epitope(s) that are localized in the kringle 5 domain of apo(a) and do not cross-react with plasminogen or the kringle 4 domain of apo(a). Such monoclonal antibodies may serve as accurate markers for the detection and diagnosis of heart disease.